Tannin-Titanium Oxide Multilayer as a Photochemically Suppressed

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Applications of Polymer, Composite, and Coating Materials

Tannin-Titanium Oxide Multilayer as a Photochemically Suppressed Ultraviolet Filter Ho Yeon Son, Bon Il Koo, Jun Bae Lee, Kyeong Rak Kim, Woojin Kim, Jihui Jang, Moung Seok Yoon, Jae-Woo Cho, and Yoon Sung Nam ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09200 • Publication Date (Web): 24 Jul 2018 Downloaded from http://pubs.acs.org on July 28, 2018

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ACS Applied Materials & Interfaces

Tannin-Titanium Oxide Multilayer as a Photochemically Suppressed Ultraviolet Filter Ho Yeon Son1,†, Bon Il Koo1,†, Jun Bae Lee2, Kyeong Rak Kim1, Woojin Kim3, Jihui Jang2, Moung Seok Yoon2, Jae-Woo Cho3, and Yoon Sung Nam1,4,* 1

Department of Materials Science and Engineering, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea E-mail: [email protected]

2

Innovation Lab, Cosmax Research & Innovation Center, 662 Sampyong-dong, Bundang-gu, Seongnam, Gyeonggi-do, 13486, Republic of Korea

3

Pathology Research Center, Department of Jeonbuk Inhalation Research, Korea Institute of Toxicology, 30 Baekhak-1-gil, Jeongup, Jeonbuk, 56212, Republic of Korea 4

KAIST Institute for the NanoCentury, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea

ABSTRACT UV filters can initiate redox reactions of oxygen and water when exposed to sunlight, generating reactive oxygen species (ROS) that deteriorate the products containing them and cause biological damages. This photochemical reactivity originates from the high chemical potential of UV filters, which also determines the optical properties desirable for sunscreen applications. We hypothesize that this dilemma can be alleviated if the photochemical pathway of UV filters is altered to coupling with redox active molecules. Here, we employ tannic acid (TA) as a key molecule for controlling the photochemical properties of titanium dioxide nanoparticles (TiO2 NPs). TA provides an unusual way for layer-by-layer assembly of TiO2 NPs by a formation of ligand-to-metal charge transfer complex which alters the nature of UV absorption of TiO2 NPs. The galloyl moieties of TA efficiently scavenge ROS due to the stabilization of ROS by intramolecular hydrogen bonding, while facilitating UV screening through direct charge injection from TA to the conduction band of TiO2. The TiO2-TA multilayers assembled in open porous

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polymer microspheres substantially increased sun protection while dramatically reducing ROS under UV exposure. The assembled structure exhibits excellent in vivo anti-UV skin protection against epidermal hyperplasia, inflammation, and keratinocyte apoptosis without long-term toxicity.

KEYWORDS Tannin; titanium dioxide; porous polymer microspheres; layer-by-layer; UV screening

INTRODUCTION

Wide bandgap semiconductors, such as titanium dioxide (TiO2) and zinc oxide, have been extensively utilized as UV screening materials for commercial products, including paints, cosmetic and personal care products, windows, automobiles, and photovoltaic cells.1-6 Although nano-sized semiconductors are favored due to their transparency and high surface area, their biological and environmental risks have become issues of public concern. Due to the high chemical potential of the generated electron-hole pairs, exposure to sunlight in the presence of water and air unavoidably makes these semiconductors produce reactive oxygen species (ROS).79

The excited electrons react with oxygen to produce superoxide anion (O2•-), and the holes

oxidize water into hydroxyl radical (•OH). Both of them are reactive enough to decompose various organic chemical compounds.10 In particular, when TiO2 nanoparticles (TiO2 NPs) in sunblock products are directly applied to the skin, the generated ROS are very harmful to the skin, causing chemical modification of biomolecules (e.g., DNA) and inducing inflammation responses,11-14 potentially leading to skin cancer.15,16 In addition, sunblock nanoparticles applied to the skin are eventually released to the environment and disturb aquatic ecosystems when they


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accumulate.17,18 Therefore, despite the immediate advantages of semiconductor nanoparticles, their long-term benefits have been challenged, and a better way to take advantage of these materials in consumer products is required. Surface coating or encapsulation of TiO2 NPs within silica matrices has been suggested to reduce the apparent concentration of ROS.19-21 Because the silica layer can retard the diffusion of ROS generated from TiO2 NPs, the ROS can be destructed before they diffuse out due to their very short half-life (~10-6 sec for O2•- and ~10-9 sec for •OH).22 However, the conformal coating of nanoparticles is very challenging, and the exposed surface of TiO2 NPs is still capable of generating ROS. A recent work even reported an unexpected increase in the ROS generated per nanoparticle from silica-coated TiO2 NPs.21 These results seem to be related to the improved dispersion stability of TiO2 NPs that has been observed in previous studies.23 Another problem with a silica coating is that the size of silica-coated TiO2 NPs is still in the sub-micron range, and so conventional filtration is not applicable for their separation from waste water, which can raise an environmental issue due to the accumulation of TiO2 NPs. The encapsulation of TiO2 NPs in micron-sized particles can be a very reasonable approach to solving this problem, because the separation of such larger particles is much easier and the dispersion of TiO2 NPs can be well maintained. However, the larger particles, when they break down under high or repeated mechanical stresses (e.g., hand rubbing on the skin), can release the encapsulated TiO2 NPs. In addition, solid particles larger than tens of micrometers also generate an unpleasant skin sensation, so they are not favorable for topical applications. In this report, to develop a new type of anti-UV coating with the suppression of ROS by redox chemistry, we introduce the layer-by-layer (LbL) self-assembly of TiO2 NPs with tannic acid (TA), which is a commercial form of tannin. TA serves as a key component for the


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conformal coating of colloidal TiO2 NPs, while controlling their redox photochemistry. TA is one of the polyphenols, which are widely found in foods including persimmons, grapes, tea, and red wine.24 It is well known that both the astringency and bitterness of red wine originate from tannins, which bind to proteins in saliva. Also, TA can be easily deposited on various materials via the coordination of metal ions (e.g., Fe, Cu, Al, and Zr) with galloyl moieties (3,4,5trihydroxyphenyl groups).25-27 The multistep metal-polyphenol coordination can form films and capsules that are useful for a broad range of applications.28 For anti-UV applications in topical formulations, we fabricated open porous poly(methyl methacrylate) (PMMA) microspheres (p-MS) as colloidal substrates for use as LbL coating of TiO2 NPs with TA. PMMA was used because it has been widely employed in cosmetic products due to its biological inertness and optical transparency. p-MS were prepared by single oil-inwater emulsification with phase separation followed by porogen extraction, as previously reported.29 TiO2 NPs were synthesized using a sol-gel method, and LbL coatings of TiO2 NPs with TA were carried out on the prepared p-MS. Optical properties, sun protection performance, and in vivo toxicity and anti-UV protective effects were examined to evaluate the feasibility of using this multilayered TA/TiO2 hybrid coating for topical anti-UV applications.

EXPERMENTAL SECTION

Materials. PMMA with a nominal molecular weight (Mw) of 50 kDa was obtained from Kimex Co., Ltd (Suwon, Republic of Korea). Titanium (IV) isopropoxide, tetrabutylammonium hydroxide 30-hydrate (Bu4NOH), poly(vinyl alcohol) (PVA, 88 % hydrolyzed, Mw 146 - 186 kDa),

poly(ethyleneglycol)-poly(propyleneglycol)-poly(ethylenegylcol)

triblock

copolymer

(Pluronic F127), tannic acid (TA), formaldehyde, hematoxylin, eosin, nitroblue tetrazolium


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(NBT), terephthalic acid, potassium superoxide (KO2), and 18-crown-6 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dichloromethane, isopropanol, and sodium hydroxide (NaOH) were obtained from Junsei Chemical (Tokyo, Japan). Pure 2-hydroxyterephthalic acid (2-HTAc) was obtained from Tokyo Chemical Industry (Tokyo, Japan). Potassium hydroxide (KOH) and dimethyl sulfoxide (DMSO) were obtained from Daejung chemicals (Gyeonggi-do, Republic of Korea). Milli-Q water was used as deionized water in all experiments.

Synthesis of TiO2 NPs. TiO2 NPs were synthesized as previously reported.23 Bu4NOH (1 g) was dissolved in propanol (46 mL) at an ambient temperature, and titanium isopropoxide (1 mL) was dissolved in propanol (10 mL). The titanium isopropoxide solution was slowly added into the Bu4NOH solution, and then boiled deionized water (232 mL) was slowly added into the mixture with magnetic stirring at 500 rpm at an ambient temperature. The mixture was transferred to an oil bath at 100 oC, and then magnetically stirred with reflux for 2 days. The synthesized TiO2 NPs (100 mL) were purified by dialysis (MWCO = 500 Da, SpectraPor) against deionized water (6 L) for 2 days.

Preparation of Porous PMMA Microspheres. A mixture of PMMA (5 g) and Pluronic F127 (5 g) was dissolved in dichloromethane (100 mL) in a beaker. The PMMA-Pluronic F127 solution was added to a 0.5 wt-% PVA solution (12 L), followed by homogenization at 5,000 rpm (PowerGen 700, Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 10 min at an ambient temperature. The oil-in-water emulsions were magnetically stirred at 250 rpm for 48 h to completely remove dichloromethane by evaporation, and then 2 L of ethanol was added to the suspension of microspheres to quickly extract the residual dichloromethane in the dispersed


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droplets into the aqueous phase. The solidified microspheres were collected by centrifugation at 1000 × g for 10 min, rinsed three times with an excess amount of deionized water, and then lyophilized.

Preparation of TiO2-Tannin Multilayered Microspheres. The prepared p-MS (5 mg) were dispersed in deionized water (0.95 mL). TA was dissolved in deionized water at a concentration of 10 mg mL-1. The TA solution (50 µL) was added into the p-MS suspension and then mixed vigorously using a vortex mixer for 5 min. TA-coated p-MS were collected by centrifugation at 900 × g for 5 min and rinsed three times with a 2:1 mixture of deionized water and ethanol. The collected TA-coated p-MS were dispersed into 1 mL deionized water and then mixed with 1 mL of 0.5 mg mL-1 TiO2 NP suspension with magnetic stirring at 250 rpm for 3 h. After incubation, the microspheres were collected by centrifugation at 900 × g for 5 min, rinsed three times with an excess amount of deionized water, and then lyophilized. For multiple layers of TA and TiO2 NPs on p-MS, tannin coatings and TiO2 NP deposition were achieved by alternately immersing the p-MS suspension into a TA solution (0.5 mg mL-1) for 5 min and a TiO2 NP suspension (0.5 mg mL-1) for 3 h, respectively, using the same procedures described above.

Characterization. To determine the attachment of TiO2 NPs on the p-MS after external mechanical forces, we dispersed the microspheres in deionized water with magnetic stirring at 250 rpm for 12 h. Then, we examined the TA-TiO2 hybrid p-MS using scanning electron microscopy (SEM, Hitachi S-4800) to determine whether the TiO2 NPs can be observed on the p-MS. The TiO2 NPs were observed using transmission electron microscope (TEM, Philips


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Tecnai F10) at an acceleration voltage of 200 kV. Powder X-ray diffraction patterns of (TA/TiO2)4-p-MS were recorded on using a Rigaku X-ray diffractometer D/MAX–2500 (40 kV, 300 mA). The morphology of (TA/TiO2)n-p-MS was examined using SEM at an acceleration voltage of 5 kV after osmium coatings for 25 sec. The amounts of TiO2 NPs on p-MS prepared by layer-by-layer coatings were quantitatively analyzed using thermogravimetry (TGA, NETZSCH, TG 209 F3) at a heating rate of 10 °C min−1 in air. The optical properties of TiO2polymer hybrid p-MS, pristine TiO2 NPs, TA-coated TiO2 NPs, and TA solution were analyzed using UV−visible absorption spectrophotometry (UV-1800, Shimadzu Corp., Kyoto, Japan). Ultraviolet photoemission spectroscopy (UPS) measurements were conducted using a He Ⅰ lamp at 21.2 eV under an applied bios of -5 V in an ultra-high vacuum (UHV) chamber with a base pressure of 10-9 Torr. Size distribution of TiO2 NPs and (TA/TiO2)4-p-MS before and after mixing in sunscreen lotion was obtained using a dynamic light scattering spectrophotometer (DLS, Otsuka ELSZ-1000). The mechanical properties of p-MS were examined under the compressional pressure using a uniforce pellet press machine (Z285889, Sigma-Aldrich, St. Louis, MO, USA).

Sun protection factor (SPF) Analysis. The sunscreen samples were prepared by hot process emulsification using the ingredients listed in Table S1. Part A was dissolved at 80 °C and then added to part B. The resulting mixture was homogenized using a T. K. Robomix® (Primix Corp., Osaka, Japan) at 6,000 rpm for 5 min. The resulting emulsions were cooled to 25 °C. The samples (1.2 mg cm-2) were applied to a sandblasted PMMA plate (47 × 47 × 1.5 mm3, HelioScreen, Creil, France) and then placed for 15 min in the dark at an ambient temperature.


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The average transmission of UV irradiation (n = 5) was measured from 290 nm to 400 nm at a step of 1 nm using a UV transmittance analyzer (Labsphere Inc., North Sutton, NH, USA).

NBT Assay. KO2 was used for drawing calibration curve of O2•-.30 To make the O2•solution, 28.2 mg of the KO2 and 105.8 mg of the 18-crown-6 were dissolved in 2 mL of degassed and deionized water and stirred for 4 h at room temperature. After stirring, the solutions were centrifuged at 13,500 rpm for 10 min. Supernatant of centrifuged solution with O2•- was serially diluted as 1/2, 1/4, and 1/8. One hundred forty µL of the diluted solutions were mixed with 140 µL of 1 mg mL-1 NBT solution and incubated at room temperature for 10 min. Absorbance of each samples was measured by Microplate reader (CLARIO star, BMG Labtech, Ortenberg, Germany) at 220 - 800 nm. Five hundred microliters of samples having 0.3 mg mL-1 TiO2 were mixed with 500 µL of 1 mg mL-1 NBT solution in 5 mL vials respectively. The vials with samples were exposed under 8 W UV lamp for 20 min. The UV exposed samples were centrifuged at 12,000 rpm for 5 min. After removing supernatants, formazans in the pellets were dissolved in 540 µL of DMSO with 460 µL of 2 M KOH.31 Absorbance of supernatant after centrifugation was measured by Microplate reader at 220 - 800 nm.

Terephthalic Acid Assay. To ensure solubility of the TAc, 1 mM of the terephthalic acid was dissolved in 4 mM of NaOH.32 Five hundred microliters of samples having 0.3 mg mL-1 TiO2 were mixed with 500 µL of 1 mM terephthalic acid solution in 5 mL vials respectively. The vials with samples were exposed under 8 W UV lamp for 20 min. Fluorescence intensity of the UV irradiated samples was measured by Microplate reader at 320 nm of excitation wavelength


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and 350 - 600 nm of emission wavelength ranges. Pure 2-HTAc was used for drawing calibration curve for calculation of •OH concentration.

In vivo Toxicity Analysis. The SKH-1 hairless mouse (Orientbio, Seongnam, Republic of Korea) was used for in vivo experiment. The mouse was treated with phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, pH 7.4), blank lotion, p-MS, TiO2 NPs, and (TA/TiO2)4-p-MS with six total applications for 3 days. For the lotion containing materials mixed with vortexing and stirring, 5 mg of TiO2 or 11.7 mg of PMMA or 16.7 mg of (TA/TiO2)4-p-MS were used per day. For all samples, a blank lotion (245 mg) was used and had a percentage of PMMA: 3.9 wt-%, TiO2: 1.7 wt-%, and (TA/TiO2)4-p-MS: 5.6 wt-%. Skin strips were harvested from the treated dorsal skin from a mouse for histological examination on 3 days after the final treatment. Formalin fixation samples were prepared by standard tissue procedure and stained by hematoxylin and eosin (H&E) and examined by a dermatologist using autostainer XL (Leica microsystems, Wetzlar, Germany). Analysis of skin images was performed using upright microscopy (eclipse Ni, Nikon, Tokyo, Japan) with slide scanning machine (aperio scanscope XT, Leica microsystems, Wetzlar, Germany).

In vivo Anti-UV Evaluation. The dorsal skin of a nude mouse was cleansed with 70 % alcohol and compartmentalized with five areas. The mouse was treated with lotions containing pMS, TiO2 NPs, and (TA/TiO2)4-p-MS, respectively. The mouse was then exposed to 8 W UVB irradiation for 1 min (2,160 J m-2). In addition, mice were also exposed to the same UV source for 1 min every day for a week for the long-term evaluation of UV effects. The remaining skin was covered with aluminium foil. Three days after the UV exposure, the dorsal skin was excised


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and fixed with formalin for histological analysis. Images for epidermal thickness were analyzed using software slide scanning machine (aperio scanscope XT, Leica microsystems, Wetzlar, Germany).

Skin penetration of TiO2. To determine the skin penetration of TiO2 NPs, we applied 1 mg of TiO2 NPs and 3.4 mg of (TA/TiO2)4-p-MS dispersed in water on the dorsal skin of mice, followed by incubation for 30 min and rinsed with PBS. The existence of TiO2 on the mouse dorsal skin was examined using SEM at an acceleration voltage of 5 kV after osmium coating for 25 sec.

γH2AX analysis. The mice were irradiated with UV irradiation for 5 min after the application of the samples to the dorsal skin. After 20 h, the epidermis was separated from the dermis by incubating the skin in a solution of 1 mg mL-1 Dispase II in PBS for 20 h. The epidermis was stained with γH2AX antibody (1 mg mL-1, clone JBW30, Millipore) for 24 h and then stained with Alexa-568-goat-anti-mouse IgG for 2 h. Finally, after mounting with DAPI (Invitrogen), images were taken using confocal fluorescence microscopy and analyzed with Image J.33

RESULTS AND DISCUSSION

The p-MS were prepared by single oil-in-water emulsification with Pluronic F127 as an extractable porogen, as described in Figure S1. The open macroporous structure was generated by the combination of phase separation and porogen leaching.34,35 PMMA and Pluronic F127


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were dissolved in dichloromethane, initially forming a homogenous phase. When the mixed polymer solution was exposed to an aqueous solution, non-solvent-induced phase separation occurred due to the influx of water into the organic droplet. Through spinodal decomposition, small fluctuations in composition tend to generate continuous biphasic domains, where a mass transfer across the boundary makes each phase purer with time.36 Interconnected cylindrical pores tend to be initially generated, followed by the formation of enlarged spheroidal pore structures via coalescence. PMMA may migrate to and stay in the polymer-rich phase, while Pluronic F127, an amphiphilic copolymer with high solubilities in both organic and water-rich phases, is expected to reside at the interface and enlarge open cellular pores when the structures are solidified as dichloromethane diffuses out to the aqueous phase and is eventually removed by evaporation. Even after the solidification, Pluronic F127, because of its high solubility in water, can diffuse from the polymer-rich droplets to the aqueous phase. Finally, stable macroporous structures of p-MS were formed when Pluronic F127 was completely leached out from the solidified PMMA microspheres. The p-MS, with a highly open porous structure, were prepared using a 1:1 weight ratio of PMMA to Pluronic F127. The size distribution of the prepared microspheres was determined from SEM images, and the average diameter of p-MS was 11.4 ± 4.1 µm (Figure S2a,b). The surface area and total pore volume of p-MS were 9.84 m2 g-1 and 0.039 cm3 g-1, respectively, as determined by Brunauer-Emmett-Teller (BET) analysis using nitrogen gas (Figure S2c). The pore volume distribution was obtained by the Barrett, Joyner, and Halenda (BJH) method, which indicated that the p-MS have macroporous structures with pore diameters greater than 50 nm (Figure S2d). To examine the mechanical properties of p-MS, we applied compressional pressures to the microspheres. The microspheres maintained their shape and structure even at an applied pressure near 2 MPa (Figure S3). Withstanding 2 MPa of


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pressure is sufficient for the microspheres to be used for cosmetic and personal care applications, since the pressure exerted on the microspheres by a gentle and soft contact with a finger is less than 10 kPa.37,38 Colloidal TiO2 NPs were synthesized by the sol-gel method. The reaction was initiated by the hydrolysis of titanium (IV) isopropoxide in a mixture of water and propanol, followed by condensation between titanium hydroxide and titanium isopropoxide. The condensation process was accelerated by the addition of tetrabutylammonium hydroxide (Bu4NOH) to form highly nucleophilic TiO-; this was followed by reflux at 100oC for two days.39 The average dimensions of the TiO2 NPs were 6.7 ± 0.7 nm in width and 129.0 ± 34.1 nm in length, as determined by TEM images (Figure 1a). High-resolution TEM analysis indicates that the d-spacing of adjacent lattice planes was 3.5 Å for the (101) plane. TiO2 NPs, with a high aspect ratio (19.3:1), were generated due to the different growth rates in the [101] and [001] directions. The growth in the [001] direction is favored as the concentration of Bu4N+ increases because of the preferred interaction of Bu4N+ with the (101) surfaces.39 The LbL self-assembly process of TiO2 NPs with TA is illustrated in Figure 1b. As for the TA coating, the prepared p-MS (5 mg) were dispersed in a TA solution (0.5 mg mL-1) at room temperature. The particle suspension was vigorously vortexed to allow TA to infiltrate the internal pores of p-MS. There was no significant change in the color or the surface roughness of the p-MS after TA coating (Figure S4). TA can be rapidly adsorbed onto the surface of the p-MS due to the high surface binding affinity of TA, which is a result of hydrogen bonding.25,40-42 In addition, residual PVA and Pluronic F127 adsorbed on the p-MS after washing can strongly interact with TA via hydrogen bonding.43 The amounts of TA adsorbed on the p-MS were about 22 µg per mg of p-MS, as determined from the changes in the absorption spectra of the TA


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solution (Figure S5). When the synthesized TiO2 NPs were added to the TA-coated p-MS, they spontaneously attached to the outer and internal surfaces of the TA-coated p-MS. The color of the p-MS changed from white to yellowish as the TA-coated p-MS were mixed with the TiO2 NP suspension (Figure S4). We speculate that the color change may be due to the formation of a ligand-to-metal charge transfer complex between the hydroxyl groups of TA and Ti4+ on the surfaces of the TiO2 NPs.44 These results indicate that TA was deposited on the p-MS by simple mixing, and that the TiO2 NPs spontaneously attached to the TA-coated p-MS through the formation of a stable charge transfer complex between TA and Ti4+ on the surfaces of the TiO2 NPs. Multilayered TiO2 NPs on p-MS were prepared by LbL deposition of TA and TiO2 NPs (denoted (TA/TiO2)n-p-MS). When the (TA/TiO2)1-p-MS were added into the TA solution (pH 3.6), partially deprotonated TA was coordinated with the TiO2 NPs in the (TA/TiO2)1-p-MS. The deposited TA was completely deprotonated in the TiO2 NP suspension at a high pH (i.e., pH 11), which facilitated the deposition of TiO2 NPs. Figure 1c provides SEM images of (TA/TiO2)n-pMS with different numbers of layers of TA and TiO2 NPs. Apparently, a larger number of TiO2 NPs were attached to both the outer and internal surfaces of p-MS with the increased number of depositions. During LbL coating, TA was coated on the surfaces of the TiO2 NPs as well as the p-MS. In addition, several galloyl groups of TA can be used to assemble TiO2 NPs with each other, resulting in significantly increased amounts of TiO2 NPs attached on the surfaces of p-MS as the number of coatings increases. When the polymer templates were removed from the (TA/TiO2)4-p-MS by calcination, the TiO2 NPs retained the macroporous structures of p-MS, which indicates that TiO2 NPs were uniformly deposited on both of the outer and internal surfaces of p-MS (Figure S6). In addition, the TiO2 NPs deposited on the p-MS stably retained their assembled structures even with external mechanical forces such as magnetic stirring and


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centrifugation (Figure S7). The result implies that the TA-TiO2 hybrid p-MS can be mixed with cosmetic formulations for sunscreen applications without any noticeable change. Thermogravimetric analysis (TGA) was carried out in air to provide quantitative information on the amount of TiO2 NPs attached on the p-MS. The TGA thermograms of p-MS and (TA/TiO2)4-p-MS are shown in Figure 2a. The content of TiO2 NPs in the (TA/TiO2)4-p-MS was 30.4 wt-% as determined by TGA, while the pristine p-MS were almost entirely removed at 800 oC with a residual weight of 1.2 wt-%. The estimated maximum content of TiO2 NPs in the (TA/TiO2)4-p-MS was about 28.6 wt-% because 10 wt-% of TiO2 NPs were used against the initial weight of p-MS for each coating. The content of TiO2 NPs determined by TGA was greater than the estimated maximum content presumably due to the loss of some TA-TiO2 hybrid p-MS during the washing steps. However, the TGA results indicate that most of the TiO2 NPs used for the assembly were successfully attached on both the internal and external surfaces of pMS. The surface area and total pore volume of the (TA/TiO2)4-p-MS were 54.15 m2 g-1 and 0.18 cm3 g-1, respectively (Figure 2b). These results indicate that the TA/TiO2 multilayered p-MS, due to the mesoporous structures formed by the deposition of high aspect ratio TiO2 NPs, have a substantially increased surface area and total pore volume compared to the p-MS. In particular, the pore volume distribution of (TA/TiO2)4-p-MS shows that large macropores over 50 nm became smaller as TiO2 NPs were deposited, while mesopores with pore diameters of about 4 nm were generated in the layer of the deposited TiO2 NPs (Figure S8). The X-ray diffraction (XRD) spectrum of (TA/TiO2)4-p-MS revealed that the synthesized TiO2 NPs used for the assembly on the p-MS had anatase structures (Figure 2c). The light absorption spectra of (TA/TiO2)n-p-MS samples with different layer numbers are shown in Figure 2d. All of the p-MS were dispersed in isopropanol because the refractive


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index of PMMA (n = 1.49) is similar to that of isopropanol (n = 1.38). (TA/TiO2)n-p-MS exhibited a high absorbance in the UV region, with peaks around 250 nm and 290 nm corresponding to the absorption of TiO2 NPs (Figure 2e), whereas no absorbance for the TiO2 NPs appeared in the pristine p-MS. In particular, the absorbance of (TA/TiO2)4-p-MS significantly increased due to the increased amounts of TiO2 NPs attached on the p-MS. The dependency of the absorbance on the number of coatings was consistent with the increased number of TiO2 NPs with the increased number of layers, as observed in the SEM images (Figure 1c). The TA coating also contributed to the highly increased absorbance of the (TA/TiO2)n-p-MS in the UV region. Figure 2e shows the UV-Vis absorption spectra of the pristine and TA-coated TiO2 NPs (0.05 mg mL-1) dispersed in deionized water and a tannic acid solution (5.8 µg mL-1, corresponding to the amounts of TA adsorbed on the 0.05 mg mL-1 of TiO2 NPs). The color of the TiO2 NP suspension changed from white-bluish to yellowish, as was the case for the (TA/TiO2)n-p-MS, due to the production of a ligand-to-metal charge transfer complex between the TiO2 NPs and TA. The absorbance measurements showed peaks around 215 nm and 274 nm for TA due to the phenolic structures of the galloyl groups in TA.32 The peak around 215 nm also appeared in the absorbance of the TA-coated TiO2 NPs, while no peak was observed for the pristine TiO2 NPs. The absorption spectra show that the increased absorbance of the TA-coated TiO2 NPs in the UV region can be attributed to the ligand-to-metal charge transfer complex rather than to a simple sum of the absorbance of TA and TiO2 NPs. To verify the increased UV absorption due to the ligand-to-metal charge transfer complex, the valence band (VB) and occupied molecular orbital electronic structures were investigated using ultraviolet photoelectron spectroscopy (UPS) measurements (Figure 2f). The VB edge of the TiO2 NPs was found at a binding energy of 3.26 eV, determined using a linear


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extrapolation of the VB edge to the background of the spectrum. The optical band gap was 3.56 eV (350 nm) for the pristine TiO2 NPs, determined by the linear extrapolation of the absorption edge to the background of the spectrum. In the UPS spectrum of TA, two low binding energy regions can be clearly distinguished; these are derived from the highest occupied molecular orbital (HOMO) and by energy states lower than the HOMO level, with band edges of 2.14 eV and 3.70 eV. The energy difference between the HOMO and the lower energy states is comparable to the energy difference between the two absorption edges of TA, 3.83 eV (324 nm) and 5.25 eV (236 nm). In particular, the two low binding energy regions of TA also appeared in the TA-coated TiO2 NPs, with band edges of 1.80 eV and 3.20 eV, without significant change of the energy difference. Consequently, the increased absorption of the TA-coated TiO2 NPs in the visible and UV regions is attributed to direct electron injection from both the HOMO and the lower energy states of TA to the conduction band of TiO2 NPs (Figure 2g).45 The energy difference between the lower energy states of TA and the conduction band of the TiO2 NPs corresponds to the optical band gap of the TA-coated TiO2 NPs, 3.54 eV (350 nm). In addition, absorption due to the intramolecular excitation of TA appears at 215 nm in the absorption spectrum of the TA-coated TiO2 NPs. Next, we investigated the suitability of (TA/TiO2)4-p-MS for UV light screening by measuring the SPF in the UV range of 290 to 400 nm. Each of the samples was prepared by adding TiO2 NPs, p-MS, and (TA/TiO2)4-p-MS, respectively, to the control sunscreen lotion. The compositions of the control formulation are listed in Table S1. The concentration of added TiO2 NPs and (TA/TiO2)4-p-MS was 0.6 wt-%, based on the weight of the TiO2 NPs. The amount of p-MS (1.4 wt-%) was also similar to that used for (TA/TiO2)4-p-MS. The measured SPF values were 31.1 ± 4.6 for the control formulation, 36.8 ± 1.4 for the sample with TiO2, 38.6 ± 3.1 for


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the sample with p-MS, and 50.0 ± 6.4 for the sample with (TA/TiO2)4-p-MS. The enhancement in the SPF value (∆ SPF) of the control lotion for the tested sunscreen materials was only 5.7 for the TiO2 NPs (Figure 2h). Interestingly, the sample containing the p-MS exhibited considerable SPF enhancement, with a ∆SPF of 7.5, probably due to light scattering caused by the porous structures of the p-MS, which can reduce the light transmission in a wide range of wavelengths. The sample containing the (TA/TiO2)4-p-MS exhibited excellent UV protection, with a significantly increased SPF value and a ∆SPF of 18.9. The synergistic SPF enhancement of the (TA/TiO2)4-p-MS resulted from a combination of the structural advantages of the p-MS and the high UV absorption of the TA-coated TiO2 NPs. In particular, the (TA/TiO2)4-p-MS can prevent the secondary aggregation of TiO2 NPs by homogeneous dispersion of the hybrid microspheres, while the TiO2 NPs were not able to effectively screen UV light due to their poor dispersion in the sunscreen sample.46 The aggregation of TiO2 NPs after mixing in the sunscreen lotion can be clearly observed in the photographs of the TiO2 NP suspension and in the size distribution, measured by dynamic light scattering (DLS) (Figure S9). The TiO2 NPs generate photoexcited electrons and holes when absorbing UV light. The electrons and holes can react with oxygen and water, respectively, to produce ROS materials such as superoxide anions and hydroxyl radicals, which cause skin aging and tissue damage (Figure 3a). TA, using its galloyl moieties, can efficiently scavenge the ROS generated from the TiO2 NPs. The antioxidant properties of the (TA/TiO2)4-p-MS were evaluated by using terephthalic acid analysis to measure the concentrations of hydroxyl radicals.47,48 The concentration of •OH was investigated by measuring the fluorescence peak around 430 nm for 2hydroxyterephthalic acid; this peak forms when terephthalic acid chemically binds with the •OH radical (Figure S10a). The pristine TiO2 NPs generated •OH radicals at a concentration of 4.64 ±


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1.03 nmol mL-1 under UV irradiation, while the (TA/TiO2)4-p-MS exhibited efficient suppression of the generation of •OH radicals at a concentration of 0.14 ± 0.02 nmol·mL-1 (Figure 3b). These results indicate that the production of •OH radicals was inhibited by about 97% or more due to the tannin layers in the (TA/TiO2)4-p-MS. To confirm the antioxidant activity of TA in the (TA/TiO2)4-p-MS, we also used NBT assay to examine the generation of superoxide anion.49,50 The concentration of superoxide anion was quantitatively analyzed according to the absorption of formazan formed by O2•- at 680 nm (Figure S10b). The concentrations of O2•- generated by the pristine TiO2 NPs and the (TA/TiO2)4-p-MS under 3.6 mW cm-2 of UVB irradiance were 73.8 ± 3.9 µmol mL-1 and 41.6 ± 1.2 µmol mL-1, respectively (Figure 3c). These results indicate that the (TA/TiO2)4-p-MS exhibited a much lower generation of O2•- (44 % of the value) compared to the TiO2 NPs. The galloyl moieties of TA can presumably capture free radicals through both the hydrogen transfer process and single electron transfer. In particular, the hydrogen atom transfer will dominantly occur due to the low bond dissociation enthalpy (BDE) of hydroxyl groups.51 Furthermore, in the galloyl moiety, the central hydroxyl group will have a lower BDE than the outer hydroxyl groups due to the stronger electronic contribution and hydrogen bond strengths by the two ortho hydroxyl groups, which can react with free radicals faster and break the chain reaction formed by free radicals.51 The radical formed in the galloyl moiety is stabilized by intra-molecular hydrogen bonding with the two ortho hydroxyl groups, as shown in Figure 3a (right panel); this process enables the efficient scavenging of free radicals generated from the TiO2 NPs.51,52 Since TA can be conformally deposited on the TiO2 NPs unlike other antioxidant nanoparticles such as CeO2 NPs, the free radicals generated from the surfaces of TiO2 NPs can be efficiently removed before they diffuse out. In addition, TA can avoid some problems shown in the antioxidant nanoparticles such as a


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severe drop in ROS scavenging activity due to the size of nanoparticles55 and has the advantage of mediating the assembly of TiO2 NPs due to its sticky properties. The long-term toxicity of the sunscreen lotion samples containing the p-MS, TiO2, and (TA/TiO2)4-p-MS was examined by applying the samples to dorsal nude mouse skin six times over a period of three days. For histological examination, all dorsal mouse skins were stained with H&E. In all the samples, no histological toxicity or inflammation was observed, and the thickness of the epidermis was normal (Figure 4a). In addition, the histological analysis of the dorsal skins revealed normal skin follicles and dermis, without any symptoms such as the presence of inflammatory infiltrates.33 Next, we investigated the UV protective ability of the TATiO2 hybrid p-MS against sunburn on mouse skin. The dorsal skin of one mouse was divided into six areas, which were treated with PBS, blank lotion, p-MS, TiO2 NPs, and (TA/TiO2)4-pMS. Three days after UVB exposure, in the histological analysis, the skin treated with (TA/TiO2)4-p-MS was found not to exhibit epidermal hyperplasia, apoptotic keratinocytes, or inflammation. However, histological photographs of the dorsal skins treated with PBS, blank lotion, p-MS, and TiO2 NPs exhibited some damage after the same UVB exposure, such as mild epidermal hyperplasia in the epidermis (Figure 4b,c, yellow arrows) and inflammation including neutrophils, granulocytes, macrophages and giant cells in the dermis or hair follicles (Figure 4b, black arrows). The epidermal hyperplasia occurred due to the increasing number of keratinocytes during the differentiation of the stem cells in basal layer via UVB exposure.55 In particular, the skin treated with TiO2 NPs exhibited the thickest epidermis of all the samples, presumably due to ROS generated from the TiO2 NPs (Figure 4c,d). It was reported that the generation of mitochondrial ROS induces the differentiation of basal epidermal cells to keratinocytes, which cause the increased epidermal thickness.56 We speculate that the ROS generated from the TiO2


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NPs by UV illumination can induce the epidermal growth, while no epidermal hyperplasia was observed in the (TA/TiO2)4-p-MS due to their great ROS scavenging ability. In addition, the severe epidermal hyperplasia in the skin treated with TiO2 NPs can be attributed to the penetration of TiO2 NPs into the skin. The amounts of TiO2 determined by ICP-MS analysis were 1.2 µg cm-2 and 4.6 ng cm-2 for the skins treated with TiO2 NPs and (TA/TiO2)4-p-MS, respectively (Figure S11). The results show that the penetration of TiO2 NPs was effectively prevented in the (TA/TiO2)4-p-MS as the TiO2 NPs strongly bound to the microspheres which hardly penetrate the skin due to their large particle size. Therefore, the (TA/TiO2)4-p-MS does not cause any potential side effects to the skin. Note that the aggregation level of the TiO2 NPs can also greatly influence their skin penetration though it is not precisely controllable. The skins treated with PBS, blank lotion, and p-MS also exhibited apoptosis of keratinocyte, which is one of the major causes of skin aging that can be induced by UVB exposure to the epidermis (Figure 4c, white circle).16 The results indicate that PBS, blank lotion, and p-MS exhibit weak UV screening effects insufficient for the protection of the skin, while the TA-TiO2 hybrid p-MS exhibited excellent UV protective ability against sunburn without any UV-induced damage. The similar results as described above were also shown in the additional experiments on the longterm effects with multiple applications of the sunscreen lotion samples containing the p-MS, TiO2 NPs, and (TA/TiO2)4-p-MS (Figure S12). To examine the UV-induced damage at the DNA level, we immuno-stained γH2AX, which is a variant of phosphorylated histone H2A recruited to the site of DNA double-strand breaks (DSBs), allowing us to identify the DSB sites in the epidermis.56 The mouse dorsal skin was treated with PBS, blank lotion, and lotions containing p-MS, TiO2 NPs, and (TA/TiO2)4-pMS, respectively, and then exposed to UV irradiation for 5 min. At 20 h after UV irradiation, the


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mice were sacrificed, and the epidermis was separated from the dermis using Dispase II. A high level of γH2AX was observed in all of the specimens except the one treated with (TA/TiO2)4-pMS (Figure 5a). More γH2AX staining images from different skin regions are added to Figure S13. Although UV does not directly induce DSBs, ROS can be generated from mitochondria by UV irradiation and induce DSBs.57,58 Accordingly, γH2AX foci can be accumulated in the epidermis unless UV is blocked properly. However, we also observed the similar level of γH2AX foci in the skin that received TiO2 NPs, providing another evidence for the toxic effects of TiO2 NPs that can efficiently generate the ROS under UV illumination. On the other hand, in the (TA/TiO2)4-p-MS treated skin, γH2AX barely existed, which indicates that UV was very effectively blocked. In addition, we counted the number of γH2AX foci using Image J and performed statistical analysis to clearly show the difference of the skin treated with (TA/TiO2)4p-MS as shown in Figure 5b.

CONCLUSION

In this study, to develop efficient and safe sunscreen materials for topical applications, we have demonstrated a facile method to immobilize tannin and TiO2 NPs in a colloidal porous structure and simultaneously generate an ROS-suppressive redox microenvironment. Using LbL selfassembly via ligand-to-metal complexation, TA and TiO2 NPs were directly deposited onto pMS. The TA-TiO2 hybrid p-MS exhibited strong absorptivity in the UV region. It was possible to precisely control the absorption intensity according to the number of LbL depositions. The antiUV effect, indicated by the SPF, of the TA-TiO2 hybrid p-MS dispersed in typical lotion formulation, was clearly higher than that of TiO2 NPs when samples were normalized to the weight of the TiO2 NPs. The increased optical cross-sectional area and better homogenous


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dispersion contributed to the increased UV filtering. Also, under UV irradiation, compared to the colloidal TiO2 NPs, the TA-TiO2 hybrid p-MS produced much lower quantities of superoxide anions and hydroxyl radicals. The presence of tannin changed the redox microenvironment of the TiO2 NPs and suppressed ROS generation. Histological examination using a mouse model showed that the hybrid had anti-UV skin protection higher than that of TiO2 NPs in categories of epidermal hyperplasia, inflammatory infiltrates, and keratinocyte apoptosis, while long-term exposure of the skin to the hybrid material was not found to lead to any significant toxicity. We anticipate that the TA-TiO2 hybrid coating, through the combined effects of dispersion stabilization and ROS suppression, might open up new routes to more efficient, safer anti-UV materials for topical applications.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.XXXX ((please add manuscript number)) Table for compositions of a control lotion formulation; illustration of the preparation procedures of p-MS; various characterization data of p-MS and (TA/TiO2)4-p-MS; fluorescence and UV-Vis absorption spectra for estimating ROS concentration; additional H&E, DAPI, and γH2AX staining images (PDF)

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] ORCID Yoon Sung Nam: 0000-0002-7302-6928 Bon Il Koo: 0000-0003-3695-7579 Kyeong Rak Kim: 0000-0001-8007-6272 Author Contributions † Ho Yeon Son and Bon Il Koo contributed equally to this work. Notes


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The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was supported by Basic Science Research Program and Nano·Material Technology Development Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2016R1A2B4013045, NRF2017M3A7B4052797, and NRF-2017M3A7B4042235).

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Figure 1. (a) TEM images of TiO2 NPs as synthesized. Scale bar: 100 nm (upper panel) and 10 nm (bottom panel) (b) Schematic illustration of the LbL self-assembly of TA and TiO2 NPs, and sample with only TA on p-MS. (c) SEM images of TA-coated p-MS and (TA/TiO2)n-p-MS. Scale bar: 2 µm (upper panels) and 200 nm (bottom panels).


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Figure 2. (a) TGA thermograms of p-MS and (TA/TiO2)4-p-MS. (b) Isotherms of N2 adsorption (inset: BET surface area plots) and (c) XRD pattern of (TA/TiO2)4-p-MS. (d) UV-Vis absorption spectra of p-MS and (TA/TiO2)n-p-MS, where n = 1 (yellow), 2 (green), 3 (blue), and 4 (red). (e) UV-Vis absorption spectra of pristine and TA-coated TiO2 NPs dispersed in deionized water (0.05 mg mL-1), and TA solution (5.8 µg mL-1). (f) UPS spectra of pristine and TA-coated TiO2 NPs, and TA. (g) Schematic energy level diagram of TiO2/TA interface. (h) In vitro SPF of lotions containing TiO2 NPs, p-MS, and (TA/TiO2)4-p-MS against the control sample with SPF value of 31.1. *p < 0.01, n = 5.


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Figure 3. (a) Schematic illustration of ROS generation from the TiO2 NPs under UV irradiation and ROS scavenging mechanisms by TA. (b) •OH and (c) O2•- concentrations of TiO2 NPs and (TA/TiO2)4-p-MS after UV irradiation. *p < 0.01.


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Figure 4. (a) Histology of dorsal mouse skin sections treated with PBS, blank lotion, and lotions containing p-MS, TiO2 NPs, and (TA/TiO2)4-p-MS. Scale bar = 100 µm. (b and c) Histology of dorsal mouse skin sections treated with the same materials and then exposed to UVB irradiation for 1 min. Evidence can be seen for epidermal hyperplasia (double-headed yellow arrow indicates the epidermal thickness), inflammatory infiltrates (black arrow), and keratinocyte apoptosis (white arrow and circle). Scale bar = 100 µm. (d) Epidermal thickness measured using microscopy software (n=10, *p < 0.01).


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Figure 5. (a) γH2AX staining of mouse dorsal epidermal sheets treated with different topical materials and irradiated by UV light for 5 min. Scale bar = 50 µm. (b) The number of γH2AX positive cells counted using Image J (n = 3, *p < 0.01).


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Tannin-Titanium Oxide Multilayer as a Photochemically Suppressed

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